A model system has been developed utilizing organotypic cultures of rat hippocampus in which a single cycle of ethanol exposure and removal results in significant "spontaneous" neuronal death, mainly in the pyramidal cells of the CA1 region. This in vitro model is potentially valuable for understanding the mechanisms underlying alcoholic brain damage, arguably the most important preventable cause of dementia in the USA. The data obtained thus far suggest that, in this model, neuronal damage is mostly excitotoxic and occurs during ethanol withdrawal. The primary hypothesis is that ethanol-induced adaptations in the glutamatergic system lead, on withdrawal of ethanol, to a cascade of changes including synaptic hyperactivity, network excitation, and excess release of endogenous glutamate and polyamines. These mediators then co-activate glutamate/NMDA receptors (NMDARs) that may be "supersensitive" to both agents, and which cause toxic Ca2+ entry. The primary specific aim is test these hypotheses and to elucidate the pre-synaptic and post-synaptic mechanisms that lead to withdrawal-induced toxicity in this model. Selective antagonists for ionotropic and metabotropic glutamate receptors and calcium channels will be used to probe mechanisms for initial synaptic excitation (calcium orange fluorescence microscopy), release of glutamate and polyamines (HPLC), distribution and sensitivity of NMDARs ([125I]MK801 autoradiography) and the relation between cumulative Ca2+ uptake (radiotracer 45Ca2+) and neurotoxicity (propidium iodide fluorescence microscopy). A second major aim is to investigate the role of network excitation in these changes by studying effects of transecting neural pathways within the culture on these parameters. Mechanisms that rely on transsynaptic activation should be prevented by this procedure. All studies in the project will focus on the regional distribution of toxicity within the slice culture, and the mechanisms that contribute to this distribution. In addition to testing the hypotheses, these experiments should suggest novel therapeutic interventions into alcohol-induced neurodegenerative disease, and elucidate why specific neuronal phenotypes are susceptible to alcohol withdrawal. Both are research priorities in the recent NIAAA Review of Neuroscience and Behavioral Science Portfolio.